Piggybac transposon variants and methods of use

ABSTRACT

The present invention provides hyperactive piggyBac transposons, in particular hyperactive piggyBac transposons from  Trichoplusia ni  (cabbage looper moth) that transpose at a higher frequency than wildtype. The invention also features integration defective piggyBac transposons. The piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell. The gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. Ser. No.13/203,393, filed Aug. 25, 2011, which is a national stage applicationfiled under 35 U.S.C. §371 of international application no.PCT/US2010/025386, filed Feb. 25, 2010, which claims the benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Application No.61/155,207, filed Feb. 25, 2009, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Typical methods for introducing DNA into a cell include DNA condensingreagents such as calcium phosphate, polyethylene glycol,lipid-containing reagents, such as liposomes, multi-lamellar vesicles,as well as virus-mediated strategies. However, such methods can havecertain limitations. For example, there are size constraints associatedwith DNA condensing reagents and virus-mediated strategies. Further, theamount of nucleic acid that can be transfected into a cell is limited invirus strategies. Not all methods facilitate insertion of the deliverednucleic acid into cellular nucleic acid, and while DNA condensingmethods and lipid-containing reagents are relatively easy to prepare,the insertion of nucleic acid into viral vectors can be labor intensive.Virus-mediated strategies can be cell-type or tissue-type specific, andthe use of virus-mediated strategies can create immunologic problemswhen used in vivo.

One suitable tool to address these issues are transposons. Transposons,or transposable elements, include a (short) nucleic acid sequence, withterminal repeat sequences upstream and downstream. Active transposonsencode enzymes that facilitate the excision and insertion of the nucleicacid into target DNA sequences.

Transposable elements represent a substantial fraction of manyeukaryotic genomes. For example, ˜50% of the human genome is derivedfrom transposable element sequences, and other genomes, for exampleplants, may consist of substantially higher proportions of transposableelement-derived DNA. Transposable elements are typically divided intotwo classes, class 1 and class 2. Class 1 is represented by theretrotransposons (LINEs, SINEs, LTRs, and ERVs). Class 2 includes the“cut-and-paste” DNA transposons, which are characterized by terminalinverted repeats (TIRs) and are mobilized by an element-encodedtransposase. Currently, 10 superfamilies of cut-and-paste DNAtransposons are recognized in eukaryotes (Feschotte and Pritham, 2007).

While class 2 elements are widespread and active in a variety ofeukaryotes, they have been thought to be transpositionally inactive inmammalian genomes. This conclusion was based primarily on the initialanalyses of the human and mouse genome sequences. While both speciesharbor a significant number and a diverse assortment of DNA transposons,they show no signs of recent activity (Lander et at. 2001; Waterston etal. 2002). For example, there are more than 300,000 DNA elementsrecognizable in the human genome, which are grouped into 120 familiesand belong to five superfamilies A large subset of these elements (40families; ˜98,000 copies) were integrated in the last 40-80 millionyears (Myr), but there remains no evidence for any human DNA transposonfamilies younger than ˜37 Myr (Pace and Feschotte, 2007).

The natural process of horizontal gene transfer can be mimicked underlaboratory conditions. In plants, transposons of the Ac/Ds and Spmfamilies have been routinely transfected into heterologous species(Osborne and Baker, 1995 Curr. Opin. Cell Biol. 7, 406-413). In animals,however, a considerable obstacle to the transfer of an active transposonsystem from one species to another has been that of species-specificityof transposition due to the requirement for factors produced by thenatural host.

Both invertebrate and vertebrate transposons hold potential fortransgenesis and insertional mutagenesis in model organisms.Particularly, the availability of alternative transposon systems in thesame species opens up new possibilities for genetic analyses.

There still remains a need for new methods for introducing DNA into acell, and particularly methods that promote the efficient insertion oftransposons of varying sizes into the nucleic acid of a cell or theinsertion of DNA into the genome of a cell while allowing more efficienttranscription/translation results than constructs as available in thestate of the art.

SUMMARY OF THE INVENTION

As described in more detail below, the piggyBac transposon fromTrichoplusia ni (cabbage looper moth) has been shown to be an activeelement in a number of insects, mice, swine and mammalian cells,including human. The present inventors have isolated Trichoplusia nipiggyBac variants that transpose at a higher frequency than wildtype.The hyperactive transposons can be used in gene transfer systems forstably introducing nucleic acids into the DNA of a cell. Moreover, thepresent inventors have identified integration defective piggyBactransposons. The gene transfer systems of the present invention can beused in methods, for example, but not limited to, gene therapy,insertional mutagenesis, or gene discovery.

Accordingly, in a first aspect, the invention features a transposoncomprising one or more hyperactive piggyBac nucleic acid sequences andvariants, derivatives and fragments thereof that retain transposonactivity.

In one embodiment, the hyperactive piggyBac transposon has a higherlevel of transposon excision compared to a wildtype piggyBac transposon.

In another embodiment, the transposon comprises 2, 3, 4, 5 or morehyperactive piggyBac nucleic acid sequences and variants, derivativesand fragments thereof that retain transposon activity.

In another further embodiment, the hyperactive piggyBac nucleic acidsequence is from the family Noctuidae. In a further related embodiment,the hyperactive piggyBac nucleic acid sequence is from the speciesTrichoplusia ni.

In another further embodiment, the nucleic acid sequence is selectedfrom SEQ ID NO: 34-SEQ ID NO: 63 or SEQ ID NO: 70 SEQ ID NO: 96.

In a further embodiment, the nucleic acid sequence encodes an amino acidsequence selected from SEQ ID NO: 3-SEQ ID NO: 32.

In another aspect, the present invention features a transposoncomprising one or more integration defective piggyBac nucleic acidsequences and variants, derivatives and fragments thereof.

In one embodiment, the integration defective piggyBac transposon has alower rate of integration as compared to a wildtype piggyBac transposon.

In another embodiment, the integration defective piggyBac nucleic acidsequence is from the family Noctuidae. In a further related embodiment,the integration defective piggyBac nucleic acid sequence is from thespecies Trichoplusia ni.

In one embodiment, the nucleic acid sequence is selected from SEQ ID NO:67-SEQ ID NO: 69.

In a further embodiment, the nucleic acid sequence encodes an amino acidsequence selected from SEQ ID NO: 64-SEQ ID NO: 66.

In another further embodiment, the wildtype piggyBac transposoncomprises a nucleic acid sequence corresponding to SEQ ID NO: 1.

In certain exemplary embodiments, the hyperactive variants comprise anamino acid change in SEQ ID NO: 2 selected from the group consisting of:G2C, Q40R, S3N, S26P, I30V, G1655, T43A, Q55R, T57A, S61R, I82V, I90V,S103P, S103T, N113S, M185L, M194V, 5230N, R281G, M282V, G316E, P410L,I426V, Q497L, K501N, K565I, N505D, S573L, 5509G, N570S, N538K, K575R,Q591P, Q591R, F594L.

In one embodiment of the above aspects, the transposon is capable ofinserting into the DNA of a cell.

In another embodiment of the above aspects, the transposon furthercomprises a marker protein.

In still another embodiment of the above aspects, the transposon isinserted in a plasmid.

In one embodiment, the transposon further comprises at least a portionof an open reading frame. In another embodiment, the transposon furthercomprises at least one expression control region. In still anotherfurther embodiment, the expression control region is selected from thegroup consisting of a promoter, an enhancer or a silencer. In anotherrelated embodiment, the transposon further comprises a promoter operablylinked to at least a portion of an open reading frame.

In one embodiment, the cell is obtained from an animal.

In another embodiment, the cell is from a vertebrate or an invertebrate.

In a further embodiment, the vertebrate is a mammal.

In one embodiment, the invention features a gene transfer systemcomprising a transposon according to any one of the above aspects; and apiggyBac transposase.

In one embodiment, the piggyBac transposase is from the familyNoctuidae. In a related embodiment, the piggyBac transposase is from thespecies Trichoplusia ni.

In a further embodiment, the piggyBac transposase comprises an aminoacid sequence corresponding to SEQ ID NO: 33.

In another embodiment, the piggyBac transposase is a mammalian piggyBactransposase.

In one embodiment, the transposon is inserted into the genome of thecell.

In another embodiment, the cell is obtained from an animal.

In another embodiment, the cell is from a vertebrate or an invertebrate.

In a further embodiment, the vertebrate is a mammal.

The present invention also features in certain embodiments a cellcomprising a transposon of any one of the above-described aspects.

In other aspects, the present invention features a pharmaceuticalcomposition comprising a transposon comprising a hyperactive piggyBacnucleic acid sequence and a piggyBac transposase, together with apharmaceutically acceptable carrier, adjuvant or vehicle.

The present invention also features a method for introducing exogenousDNA into a cell comprising contacting the cell with the gene transfersystem of the above-described aspects, thereby introducing exogenous DNAinto a cell.

In one embodiment, the cell is a stem cell.

The present invention also features a kit comprising: a transposoncomprising a hyperactive piggyBac nucleic acid sequence and instructionsfor introducing DNA into a cell.

In one embodiment, the hyperactive piggyBac nucleic acid sequence isfrom the family Noctuidae. In a further embodiment, the hyperactivepiggyBac nucleic acid sequence is from the species Trichoplusia ni.

In another embodiment, the nucleic acid is sequence selected from SEQ IDNO: 34-SEQ ID NO: 63 or SEQ ID NO: 70-SEQ ID NO: 96.

In another aspect, the present invention also features a kit comprising:a transposon comprising a integration defective piggyBac nucleic acidsequence and instructions for use.

In one embodiment, the nucleic acid sequence is selected from SEQ ID NO:67-SEQ ID NO: 69.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

DEFINITIONS

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-3,4-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

As used herein, the following terms have the meanings ascribed to thembelow, unless specified otherwise.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

As used herein, the term “integration defective” is meant to refer to atransposon that integrates at a lower frequency into the host genomethan a corresponding wild type transposon. In certain exemplaryembodiments, the inventive transposons integrate by conventionalintegration mechanisms.

As used herein, the term “nucleotide” or “polynucleotide” is meant torefer to both double- and single-stranded DNA and RNA, and combinationsthereof. A polynucleotide may include nucleotide sequences havingdifferent functions, including for instance coding sequences, andnon-coding sequences such as regulatory sequences. A polynucleotide canbe obtained directly from a natural source, or can be prepared with theaid of recombinant, enzymatic, or chemical techniques. A polynucleotidecan be linear or circular in topology. A polynucleotide can be, forexample, a portion of a vector, or a fragment. A “coding sequence” or a“coding region” is a polynucleotide that encodes a polypeptide and, whenplaced under the control of appropriate regulatory sequences, expressesthe encoded polypeptide. The boundaries of a coding region are generallydetermined by a translational start codon at its 5′ end and atranslational stop codon at its 3′ end. A regulatory sequence is anucleotide sequence that regulates expression of a coding region towhich it is operably linked. Non-limiting examples of regulatorysequences include promoters, transcriptional initiation sites,translational start sites, translational stop sites, transcriptionalterminators (including, for instance, poly-adenylation signals), andintervening sequences (introns).

As used herein, the term “operably linked” is meant to refer anucleotide sequence that is placed in a functional relationship withanother nucleotide sequence. For example, if a coding sequence isoperably linked to a promoter sequence, this generally means that thepromoter may promote transcription of the coding sequence. Operablylinked means that the DNA sequences being linked are typicallycontiguous and, where necessary join two protein coding regions,contiguous and in reading frame. Since enhancers may function whenseparated from the promoter by several kilobases and intron sequencesmay be of variable lengths, some nucleotide sequences may be operablylinked but not contiguous.

As used herein, the term “polypeptide” is meant to refer to a polymer ofamino acids of any length. Thus, for example, the terms peptide,oligopeptide, protein, antibody, and enzyme are included within thedefinition of polypeptide. This term also includes post-expressionmodifications of the polypeptide, for example, glycosylations (e.g., theaddition of a saccharide), acetylations, phosphorylations and the like.

As used herein, the term “transposon” or “transposable element” is meantto refer to a polynucleotide that is able to excise from a donorpolynucleotide, for instance, a vector, and integrate into a targetsite, for instance, a cell's genomic or extrachromosomal DNA. Atransposon includes a polynucleotide that includes a nucleic acidsequence flanked by cis-acting nucleotide sequences on the termini ofthe transposon. A nucleic acid sequence is “flanked by” cis-actingnucleotide sequences if at least one cis-acting nucleotide sequence ispositioned 5′ to the nucleic acid sequence, and at least one cis-actingnucleotide sequence is positioned 3′ to the nucleic acid sequence.Cis-acting nucleotide sequences include at least one inverted repeat(also referred to herein as an inverted terminal repeat, or ITR) at eachend of the transposon, to which a transposase, preferably a member ofthe mammalian piggyBac family of transposases, binds. In certainpreferred embodiments, the transposon is from the family Noctuidae. Infurther preferred embodiments, the transposon is a Trichoplusia ni(Cabbage looper moth) piggyBac transposon.

As used herein “Trichoplusia ni” is meant to refer to a member of themoth family Noctuidae.

An “isolated” polypeptide or polynucleotide means a polypeptide orpolynucleotide that has been either removed from its naturalenvironment, produced using recombinant techniques, or chemically orenzymatically synthesized. Preferably, a polypeptide or polynucleotideof this invention is purified, i.e., essentially free from any otherpolypeptide or polynucleotide and associated cellular products or otherimpurities.

As used herein, the term “transposase” is meant to refer to apolypeptide that catalyzes the excision of a transposon from a donorpolynucleotide (e.g., a vector) and the subsequent integration of thetransposon into the genomic or extrachromosomal DNA of a target cell.Preferably, the transposase binds an inverted sequence or a directrepeat. The transposase may be present as a polypeptide. Alternatively,the transposase is present as a polynucleotide that includes a codingsequence encoding a transposase. The polynucleotide can be RNA, forinstance an mRNA encoding the transposase, or DNA, for instance a codingsequence encoding the transposase. When the transposase is present as acoding sequence encoding the transposase, in some aspects of theinvention the coding sequence may be present on the same vector thatincludes the transposon, i.e., in cis. In other aspects of theinvention, the transposase coding sequence may be present on a secondvector, i.e., in trans. In certain preferred embodiments, thetransposase is a mammalian piggyBac transposase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Table (Table 2) that shows the amino acid changes and foldincrease in transposition from that of wildtype (normalized to 1) ininventive hyperactive variants.

FIGS. 2A1-2A2 and FIG. 2B show various transposon sequences. FIG. 2A-1shows the nucleic acid sequence of the wild type Trichoplusia nitransposon, corresponding to SEQ ID NO: 1. The corresponding amino acidsequence (SEQ ID NO: 2) is shown in FIG. 2A-2. FIG. 2B shows the aminoacid sequence corresponding to the wild type Trichoplusia nitransposase, corresponding to SEQ ID NO: 33.

FIG. 3 shows identification of excision hyperactive piggyBac mutants.FIG. 3 discloses “GLESCN” as SEQ ID NO: 130 and “WLESCN” as SEQ ID NO:128.

FIG. 4 shows the amino acid sequences of the integration defectivepiggyBac transposons, corresponding to SEQ ID NOs 64-66.

DETAILED DESCRIPTION

The ability to use a transposon for genome engineering is highlydependent upon the frequency with which it can move. For example if 1progeny in 10 has a transposon event, it will be much easier to isolatederivatives of the desired type than if the transposition event occursin 1 in 1000 progeny. The present inventors have isolated hyperactivepiggyBac transposons from Trichoplusia ni (cabbage looper moth) thattranspose at a higher frequency than wildtype. Transposons such aspiggyBac are widely used for genome engineering by insertionalmutagenesis and transgenesis in a wide variety of organisms. ThepiggyBac transposon from Trichoplusia ni has been shown to be an activeelement in a number of insect, mice, swine and mammalian cells includinghuman.

Accordingly, the present invention features hyperactive piggyBactransposons. A hyperactive piggyBac transposon is meant to refer to atransposon that has a transposon event at a higher frequency than wildtype piggyBac transposon. For example, in certain exemplary embodiments,in a hyperactive piggyBac transposon transposition occurs 0.5 fold, 1fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 25 fold, 30 fold, 40 fold, 45 fold, 50 fold ormore.

According to certain preferred embodiments of the present invention, ahyperactive piggyBac transposon is bound by a transposase, contains apair of repeat sequences. In certain preferred embodiments, the firstrepeat is typically located upstream to the nucleic acid sequence andthe second repeat is typically located downstream of the nucleic acidsequence. Accordingly, the second repeat represents the same sequence asthe first repeat, but shows an opposite reading direction as comparedwith the first repeat (5′ and 3′ ends of the complementary double strandsequences are exchanged). These repeats are then termed “invertedrepeats” (IRs), due to the fact that both repeats are just inverselyrepeated sequences. In certain embodiments, repeats may occur in amultiple number upstream and downstream of the above mentioned nucleicacid sequence. Preferably, the number of repeats located upstream anddownstream of the above mentioned nucleic acid sequence is identical. Incertain embodiments, the repeats are short, between 10-20 base pairs,and preferably 15 base pairs.

The repeats (IRs) as described herein preferably flank a nucleic acidsequence which is inserted into the DNA of a cell. The nucleic acidsequence can include all or part of an open reading frame of a gene(i.e., that part of a protein encoding gene), one or more expressioncontrol sequences (i.e., regulatory regions in nucleic acid) alone ortogether with all or part of an open reading frame. Preferred expressioncontrol sequences include, but are not limited to promoters, enhancers,border control elements, locus-control regions or silencers. In apreferred embodiment, the nucleic acid sequence comprises a promoteroperably linked to at least a portion of an open reading frame.According to certain preferred embodiments, hyperactive transposons ofthe present invention can preferably occur as a linear transposons(extending from the 5′ end to the 3′ end, by convention) that can beused as a linear fragment or circularized, for example in a plasmid.

The present invention features hyperactive piggyBac nucleic acidsequence and variants, derivatives and fragments thereof that retaintransposon activity. In preferred embodiments of the invention, thehyperactive piggyBac transposon has a higher level of transposonexcision compared to a wildtype piggyBac transposon

In certain preferred embodiments, the hyperactive piggyBac transposonnucleic acid sequence is from the family Noctuidae. In further exemplaryembodiments, the hyperactive piggyBac transposon nucleic acid sequenceis from Trichoplusia ni.

Preferred embodiments of the present invention refer to nucleic acidsencoding a hyperactive piggyBac transposon as defined herein.

It will be understood by a skilled person that numerous differentpolynucleotides can encode the same polypeptide as a result of thedegeneracy of the genetic code. In addition, it is to be understood thatskilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the polypeptide sequence encoded by thepolynucleotides used in the invention to reflect the codon usage of anyparticular host organism in which the polypeptides are to be expressed.The polynucleotides may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of the polynucleotides of the invention.

Polynucleotides such as DNA polynucleotides may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the lipid targeting sequencewhich it is desired to clone, bringing the primers into contact withmRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

It will be appreciated that the polynucleotide of the invention maycontain only coding regions. However, it is preferred if thepolynucleotide further comprises, in operable linkage, a portion ofnucleic acid that allows for efficient translation of the codingsequence. It is further preferred if the polynucleotide (when in a DNAform) further comprises a promoter in operable linkage which allows forthe transcription of the coding region and the portion of nucleic acidthat allows for efficient translation of the coding region in a targetcell.

Nucleic acids according to the present invention typically compriseribonucleic acids, including mRNA, DNA, cDNA, chromosomal DNA,extrachromosomal DNA, plasmid DNA, viral DNA or RNA. In certainpreferred embodiments, a nucleic acid is preferably selected from anynucleic sequence encoding the same amino acid sequence of a hyperactivepiggyBac transposon due to degeneration of its genetic code. Thesealternative nucleic acid sequences may lead to an improved expression ofthe encoded fusion protein in a selected host organism. Tables forappropriately adjusting a nucleic acid sequence are known to a skilledperson. Preparation and purification of such nucleic acids and/orderivatives are usually carried out by standard procedures (see Sambrooket al. 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.). Other variants of these native nucleic acids may have one or morecodon(s) inserted, deleted and/or substituted as compared to nativenucleic acid sequences. These sequence variants preferably lead tointegration defective and/or hyperactive piggyBac transposon proteinshaving at least one amino acid substituted, deleted and/or inserted ascompared to the native nucleic acid sequences of transposons. Therefore,nucleic acid sequences of the present invention may code for modified(non-natural) transposon sequences. Further, promoters or otherexpression control regions can be operably linked with the nucleic acidsencoding the proteins described herein to regulate expression of theprotein in a quantitative or in a tissue-specific manner.

In a particular embodiment, the Tichoplusia ni wildtype piggyBactransposon comprises a nucleic acid sequence corresponding to SEQ ID NO:1.

SEQ ID NO: 1 >Trichoplusia ni piggyBac transposonCCCTAGAAAGATAGTCTGCCTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCCGTCACTGTGCGTTTAGGACATCTCAGTCGCCGCTTGGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAAATGACACATGATTATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTACATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATATATATATTTTCTTGTTATAGATATCGTGACTAATATATAATAAAATGGGTAGTTCTTTAGACGATGAGCATATCCTCTCTGCTCTTCTGCAAAGCGATGACGAGCTTGTTGGTGAGGATTCTGACAGTGAAATATCAGATCACGTAAGTGAAGATGACGTCCAGAGCGATACAGAAGAAGCGTTTATAGATGAGGTACAGGAAGTGCAGCCAACGTCAAGCGGTAGTGAAATATTAGACGAACAAAATGTTATTGAACAACCAGGTTCTTCATTGGCTTCTAACAAAATCTTGACCTTGCCACAGAGGACTATTAGAGGTAAGAATAAACATTGTTGGTCAACTTCAAAGTCCACGAGGCGTAGCCGAGTCTCTGCACTGAATCATGTCAGATCTCAAAGAGGTCCGACGCGTATGTGCCGCAATATATATGACCCACTTTTATGCTTCAAACTATTTTTTACTGATGAGATAATTTCGGAAATTGTAAAATGGACAAATGCTGAGATATCATTGAAACGTCGGGAATCTATGACAGGTGCTACATTTCGTGACACGAATGAAGATGAAATCTATGCTTTCTTTAGTATTCTGGTAACGACAGCAGTGAGAAAAGATAACCACATGTCCACAGATGACCTCTTTGATCGATCTTTGTCAATGGTGTACGTCTCTGTAATGAGTGGTGATCGTTTTGATTTTTTGATACGATGTCTTAGAATGGATGACAAAAGTATACGGCCCACACTTCGAGAAAACGATGTATTTACTCCTGTTAGAAAAATATGGGATCTCTTTATCCATCAGTGCATACAAAATTACACTCCAGGGGCTCATTTGACCATAGATGAACAGTTACTTGGTTTTAGAGGACGGTGTGCGTTTAGGATGTATATGCCAAACAAGGCAAGTAAGTATGGAATAAAAATCGTCATGATGTGTGAGAGTGGTACAAAGTATATGATAAATGGAATGCCTTATTAGGGAAGAGGAACACAGACCAACGGAGCACCACTCGGTGAATACTACGTGAAGGAGTTATTAAAGCCTGTGCACGTAGTTTGTCGCAATATTACGTGTGACAATTGGTTCACCTCAATCCCTTTGGCAAAAAACTTACTACAAGAACCGTATAAGTTAACCATTGTGGGAACCGTGCGATCAAACAAACGCGAGATACCGGAAGTACTGAAAAACAGTCGCTCCAGGCCAGTGGGAACATCGATGTTTTGTTTTGACGGACCCCTTACTCTCGTCTCATATAAACCGAAGCCAGCTAAGATGGTATACTTATTATCATCTTGTGATGAGGATGCTTCTATCAACGAAAGTACCGGTAAACCGCAAATGGTTATGTATTATAATCAAACTAAAGGCGGAGTGGACACGCTAGACCAAATGTGTTCTGTGATGACCTGCAGTAGGAAGACGAATAGGTGGCCTATGGCATTATTGTACGGAATGATAAACATTGCCTGCATAAATTCTTTTATTATATACAGCCATAATGTCAGTAGCAAGGGAGAAAAGGTTCAAAGTCGCAAAAAATTTATGAGAAACCTTTACATGAGCCTGACGTCATCGTTTATGCGTAAGCGTTTAGAAGCTCCTACTTTGAAGAGATATTCGCGCGATAATATCTCTAATATTTTGCCAAATGAAGTGCCTGGTACATCAGATGACAGTACTGAAGAGCCAGTAACGAAAAAACGTACTTACTGTACTTACTGCCCCTCTAAAATAAGGCGAAAGGCAAAGCATCGTGCAAAAAATGCAAAAAAGTTTATTTGTCGAGAGCATAATATTGATATGTGCCAAAGTTGTTTCTGACTGACTAATAAGTATAATTTGTTTCTATTATGTATAAGTTAAGCTAATTACTTATTTTATAATACAACATGAGTGTTTTTAAAGTACAAAATAAGTTTATTTTAGTAAAGGAGAGAATGTTTAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATAGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTAGGATATTATCTTTAAGGTACGTCACAATATGATTATCTTTCTAGGG

In further embodiments, the Tichoplusia ni wildtype piggyBac transposonamino acid sequence corresponds to SEQ ID NO: 2, shown below:

MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF.

As described herein, in certain embodiments, the present inventionfeatures integration defective piggyBac transposons. Integrationdefective is meant to refer to a transposon that integrates at a lowerfrequency into the host genome than a corresponding wild typetransposon. In certain exemplary embodiments, the inventive transposonsintegrate by conventional integration mechanisms.

Integration defective piggyBac transposons, in certain exemplaryembodiments, are derived from the wildtype piggyBac sequence, SEQ ID NO:2. In exemplary embodiments, the integration defective piggyBactransposon comprises a change in SEQ ID NO: 2 selected from R372A orK375A.

In certain preferred embodiments, the integration defective piggyBactransposon comprises am amino acid sequence selected from SEQ ID NO: 64,SEQ ID NO: 65 or SEQ ID NO: 66.

In certain embodiments, the amino acid change in SEQ ID NO: 2 comprisesR372A and corresponds to SEQ ID NO: 64.

SEQ ID NO: 64 MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVASNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

The integration defective variant encoded by SEQ ID NO: 64 correspondsto a nucleotide change of CGA to GCA in SEQ ID NO: 1, and corresponds toSEQ ID NO: 67.

In other certain embodiments, the amino acid change in SEQ ID NO: 2comprises K375A and corresponds to SEQ ID NO: 65.

SEQ ID NO: 65 MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

The integration defective variant encoded by SEQ ID NO: 65 correspondsto a nucleotide change of AAA to GCA in SEQ ID NO: 1, and corresponds toSEQ ID NO: 68.

In other certain embodiments, the amino acid change in SEQ ID NO: 2comprises R372A, K375A and corresponds to SEQ ID NO: 66.

SEQ ID NO: 66 MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

The integration defective variant encoded by SEQ ID NO: 66 correspondsto a nucleotide change of CGA to GCA/AAA to GCA in SEQ ID NO: 1, andcorresponds to SEQ ID NO: 69.

As described herein, the present invention also features hyperactivepiggyBac transposons.

In certain preferred embodiments, the hyperactive piggyBac transposonsare generated from the integration defective piggyBac variants. That is,alterations, preferably one or more mutations, are made in theintegration defective piggyBac transposon sequence. In otherembodiments, the hyperactive piggyBac transposons are generated from thewildtype sequences. That is, alterations, preferably one or moremutations, are made in the wild type piggyBac transposon sequence.

In exemplary embodiments, the hyperactive piggyBac comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO: 3-SEQ IDNO: 32.

In other exemplary embodiments, the hyperactive piggyBac comprises anucleic acid sequence selected from the group consisting of: SEQ ID NO:34-SEQ ID NO: 63.

The hyperactive piggyBac can preferably comprise one or more nucleicacid sequences selected from the group consisting of: SEQ ID NO: 34-SEQID NO: 63. For example, the hyperactive piggyBac can may preferablycomprise 1, 2, 3, 4, 5 or more nucleic acid sequences selected from thegroup consisting of: SEQ ID NO: 34-SEQ ID NO: 63.

In certain exemplary embodiments, the hyperactive variants comprise anamino acid change in SEQ ID NO: 2 selected from the group consisting of:L15P, D19N/F395L, S31P/T164A, H33Y, E44K/K334R, E45G, C97R/T242I, S103P,R189K/G120G, R189R/D450N/R526R, M194T, M194V, S213S/V436I, I221T, S373P,N384T, 453S/N571S, T560A, N571S, S573A, S584P, M589V, M589V/D170D,S592G, F594L, Stop/WLESCN (“WLESCN” disclosed as SEQ ID NO: 128),Stop595ELESCN/H33H (“ELESCN” disclosed as SEQ ID NO: 129).

In certain exemplary embodiments, the hyperactive variants comprise anamino acid change in SEQ ID NO: 64 or 65 selected from the groupconsisting of: L15P, D19N/F395L, S31P/T164A, H33Y, E44K/K334R, E45G,C97R/T242I, S103P, R189K/G120G, R189R/D450N/R526R, M194T, M194V,S213SN436I, I221T, S373P, N384T, 453S/N571S, T560A, N571S, S573A, S584P,M589V, M589V/D170D, S592G, F594L, Stop/WLESCN (“WLESCN” disclosed as SEQID NO: 128), Stop595ELESCN/H33H (“ELESCN” disclosed as SEQ ID NO: 129).

In certain exemplary embodiments, the hyperactive variants comprise anamino acid change in SEQ ID NO: 2 selected from the group consisting of:G2C, Q40R, S3N, S26P, I30V, G165S, T43A, Q55R, T57A, S61R, I82V, I90V,S103P, S103T, N113S, M185L, M194V, S230N, R281G, M282V, G316E, P410L,I426V, Q497L, K501N, K565I, N505D, S573L, 5509G, N570S, N538K, K575R,Q591P, Q591R, F594L.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises L15P and corresponds to SEQ ID NOS 3 & 97. The hyperactivevariants encoding SEQ ID NOS 3 & 97 corresponds to a nucleotide changeof CUG to CCG in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 34or 70.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises D19N/F395L and corresponds to SEQ ID NOS 4 & 98. Thehyperactive variants encoding SEQ ID NOS 4 & 98 corresponds to anucleotide change of GAC to AAC/UUU to CUU in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 35 or 71.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S31P/T164A and corresponds to SEQ ID NOS 5 & 99. Thehyperactive variants encoding SEQ ID NOS 5 & 99 corresponds to anucleotide change of UCA to CCA/ACA to GCA in SEQ ID NOS 67 or 68 andcorresponds to SEQ ID NOS 36 or 72.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises H33Y and corresponds to SEQ ID NOS 6 & 100. The hyperactivevariants encoding SEQ ID NOS 6 & 100 corresponds to a nucleotide changeof CAC to UAC in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 37or 73.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises E44K/K334R and corresponds to SEQ ID NOS 7 & 101. Thehyperactive variants encoding SEQ ID NOS 7 & 101 corresponds to anucleotide change of GAA to AAA/AAG to AGG in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 38 or 74.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises E45G and corresponds to SEQ ID NOS 8 & 102. The hyperactivevariants encoding SEQ ID NOS 8 & 102 corresponds to a nucleotide changeof GAA to GGA in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 39or 75.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises C97R/T242I and corresponds to SEQ ID NOS 9 & 103. Thehyperactive variants encoding SEQ ID NOS 9 & 103 corresponds to anucleotide change of UGU to CGU/ACU to AUU in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 40 or 76.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S103P and corresponds to SEQ ID NOS 10 & 104. The hyperactivevariants encoding SEQ ID NOS 10 & 104 corresponds to a nucleotide changeof UCC to CCC in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 41or 77.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises R189K/G120G and corresponds to SEQ ID NOS 11 & 105. Thehyperactive variants encoding SEQ ID NOS 11 & 105 corresponds to anucleotide change of AGA to AAA/GGU to GGC in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 42 or 78.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises R189R/D450N/R526R and corresponds to SEQ ID NOS 12 & 106. Thehyperactive variants encoding SEQ ID NOS 12 & 106 corresponds to anucleotide change of AGA to AGG/GAC to AAC in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 43 or 79.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises M194T and corresponds to SEQ ID NOS 13 & 107. The hyperactivevariants encoding SEQ ID NOS 13 & 107 corresponds to a nucleotide changeof AUG to ACG in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 44or 80.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises M194V and corresponds to SEQ ID NOS 14 & 108. The hyperactivevariants encoding SEQ ID NOS 14 & 108 corresponds to a nucleotide changeof AUG to GUG in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 45or 81.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S2135/V436I and corresponds to SEQ ID NOS 15 & 109. Thehyperactive variants encoding SEQ ID NOS 15 & 109 corresponds to anucleotide change of AGU to AGC in SEQ ID NOS 67 or 68, and correspondsto SEQ ID NOS 46 or 82.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises I221T and corresponds to SEQ ID NOS 16 & 110. The hyperactivevariants encoding SEQ ID NOS 16 & 110 corresponds to a nucleotide changeof AUA to ACA in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 47or 83.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S373P between M6+ and corresponds to SEQ ID NOS 17 & 111. Thehyperactive variants encoding SEQ ID NOS 17 & 111 corresponds to anucleotide change of UCA to CCA in SEQ ID NOS 67 or 68, and correspondsto SEQ ID NOS 48 or 84.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises N384T and corresponds to SEQ ID NOS 18 & 112. The hyperactivevariants encoding SEQ ID NOS 18 & 112 corresponds to a nucleotide changeof AAC to ACC in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 49or 85.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises C453S/N571S and corresponds to SEQ ID NOS 19 & 113. Thehyperactive variants encoding SEQ ID NOS 19 & 113 corresponds to anucleotide change of UGU to AGU/AAU to AGU in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 50 or 86.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises T560A and corresponds to SEQ ID NOS 20 & 114. The hyperactivevariants encoding SEQ ID NOS 20 & 114 corresponds to a nucleotide changeof ACU to GCU in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 51or 87.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises N571S and corresponds to SEQ ID NOS 21 & 115. The hyperactivevariants encoding SEQ ID NOS 21 & 115 corresponds to a nucleotide changeof AAU to AAG in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 52or 88.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S573A and corresponds to SEQ ID NOS 22 & 116. The hyperactivevariants encoding SEQ ID NOS 22 & 116 corresponds to a nucleotide changeof UCG to GCG in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 53or 89.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S584P and corresponds to SEQ ID NOS 23 & 117. The hyperactivevariants encoding SEQ ID NOS 23 & 117 corresponds to a nucleotide changeof UCU to CCU in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 54or 90.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises M589V and corresponds to SEQ ID NOS 24 & 118. The hyperactivevariants encoding SEQ ID NOS 24 & 118 corresponds to a nucleotide changeof AUG to GUG in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 55or 91.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises M589V/D170D and corresponds to SEQ ID NOS 25 & 119. Thehyperactive variants encoding SEQ ID NOS 25 & 119 corresponds to anucleotide change of ATG to GUG/GAC to GAU in SEQ ID NOS 67 or 68, andcorresponds to SEQ ID NOS 56 or 92.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises S592G and corresponds to SEQ ID NOS 26 & 120. The hyperactivevariants encoding SEQ ID NOS 26 & 120 corresponds to a nucleotide changeof AGU to GGU in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 57or 93.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises F594L and corresponds to SEQ ID NOS 27 & 121. The hyperactivevariants encoding SEQ ID NOS 27 & 121 corresponds to a nucleotide changeof UUC to TTA in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 58or 94.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises Stop/WLESCN (“WLESCN” disclosed as SEQ ID NO: 128) andcorresponds to SEQ ID NOS 28 & 122. The hyperactive variants encodingSEQ ID NOS 28 & 122 corresponds to a nucleotide change of TGA to TGG inSEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 59 or 95.

In certain embodiments, the amino acid change in SEQ ID NOS 64 or 65comprises Stop595ELESCN/H33H (“ELESCN” disclosed as SEQ ID NO: 129) andcorresponds to SEQ ID NOS 29 & 123. The hyperactive variants encodingSEQ ID NOS 29 & 123 corresponds to a nucleotide change of TGA to GGA/CACto CAU in SEQ ID NOS 67 or 68, and corresponds to SEQ ID NOS 60 or 96.

In another preferred embodiment the nucleic acid encoding thehyperactive piggyBac transposon is selected from a nucleic acid sequenceencoding the hyperactive piggyBac transposon as defined above and beingcapable of hybridizing to a complement of a nucleic acid sequence asdefined above under stringent conditions. In another preferredembodiment the nucleic acid encoding the hyperactive piggyBac transposonis selected from a nucleic acid sequence encoding the hyperactivepiggyBac transposase as defined above and being capable of hybridizingto a complement of a nucleic acid sequence as defined above understringent conditions. Stringent conditions are, for example: 30% (v/v)formamide in 0.5*SSC, 0.1% (w/v) SDS at 42 C for 7 hours.

Assays for measuring the excision of a transposon from a vector, theintegration of a transposon into the genomic or extrachromosomal DNA ofa cell, and the ability of transposase to bind to an inverted repeat aredescribed herein and are known to the art (see, for instance, (Ivics etal. Cell, 91, 501-510 (1997); WO 98/40510 (Hackett et al.); WO 99/25817(Hackett et al.), WO 00/68399 (Mclvor et al.), incorporated by referencein their entireties herein. For purposes of determining the frequency oftransposition of a transposon of the present invention, the activity ofthe baseline transposon is normalized to 100%, and the relative activityof the transposon of the present invention determined. Preferably, atransposon of the present invention transposes at a frequency that is,in increasing order of preference, at least about 50%, at least about100%, at least about 200%, most preferably, at least about 300% greaterthan a baseline transposon. Preferably, both transposons (i.e., thebaseline transposon and the transposon being tested) are flanked by thesame nucleotide sequence in the vector containing the transposons.

The invention also features protein sequence showing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and most preferably at least 98% sequenceidentity with the protein sequence of any one of SEQ ID NOs 3-32.

The invention also features protein sequence showing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and most preferably at least 98% sequenceidentity with the protein sequence of any one of SEQ ID NOs 3-32.

The term “identity” is understood as the degree of identity between twoor more proteins, nucleic acids, etc., which may be determined bycomparing these sequences using known methods such as computer basedsequence alignments (basic local alignment search tool, S. F. Altschulet al., J. Mol. Biol. 215 (1990), 403-410). Such methods include withoutbeing limited thereto the GAG programme, including GAP (Devereux, J., etal., Nucleic Acids Research 12 (12): 287 (1984); Genetics Computer GroupUniversity of Wisconsin, Madison, (WI)); BLASTP or BLASTN, and FASTA(Altschul, S., et al., J. Mol. Biol. 215:403-410) (1999)). Additionally,the Smith Waterman-algorithm may be used to determine the degree ofidentity between two sequences.

Functional derivatives according to the present invention preferablymaintain the biological function of the mammalian transposase, i.e. thetransposase activity, the excision of the nucleic acid sequence and itsinsertion activity concerning the excised sequences into specific targetsequences. Functional derivatives according to the present invention maycomprise one or more amino acid insertion(s), deletion(s) and/orsubstitution(s) of the hyperactive variants as described herein, forexample, as those corresponding to SEQ ID NOs 3-32.

Amino acid substitutions as described herein are preferably conservativeamino acid substitutions, which do not alter the biological activity ofthe transposon or transposase protein. Conservative amino acidsubstitutions are characterized in that an amino acid belonging to agroup of amino acids having a particular size or characteristic can besubstituted for another amino acid, particularly in regions of theinventive protein that are not associated with catalytic activity or DNAbinding activity, for example. Other amino acid sequences may include,for example, amino acid sequences containing conservative changes thatdo not significantly alter the activity or binding characteristics ofthe resulting transposase. Substitutions for an amino acid sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. The polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Such alterations are not expected tosubstantially affect apparent molecular weight as determined bypolyacrylamide gel electrophoresis or isoelectric point. Particularlypreferred conservative substitutions include, but are not limited to,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free NH₂.

Amino acid insertions and substitutions are preferably carried out atthose sequence positions of that do not alter the spatial structure orwhich relate to the catalytic center or binding region of the piggyBactransposon or transposase. A change of a spatial structure byinsertion(s) or deletion(s) can be detected readily with the aid of, forexample, CD spectra (circular dichroism spectra) (Urry, 1985,Absorption, circular Dichroism and ORD of Polypeptides, in: ModernPhysical Methods in Biochemistry, Neuberger et al. (Ed.), Elsevier,Amsterdam). Suitable methods for generating proteins with amino acidsequences which contain substitutions in comparison with the nativesequence(s) are disclosed for example in the publications U.S. Pat. No.4,737,462, U.S. Pat. No. 4,588,585, U.S. Pat. No. 4,959,314, U.S. Pat.No. 5,116,943, U.S. Pat. No. 4,879,111 and U.S. Pat. No. 5,017,691,incorporated by reference in their entireties herein. Other functionalderivatives may be additionally stabilized in order to avoidphysiological degradation. Such stabilization may be obtained bystabilizing the protein backbone by a substitution of by stabilizing theprotein backbone by substitution of the amide-type bond, for examplealso by employing [beta]-amino acids.

According to certain preferred embodiments of the present invention, thetransposon of the present invention may further comprise a markerprotein. For example, in certain preferred embodiments, the nucleic acidsequence can be of any variety of recombinant proteins, e.g. any proteinknown in the art. e.g. the protein encoded by the nucleic acid sequencecan be a marker protein such as green fluorescent protein (GFP), theblue fluorescent protein (BFP), the photo activatable-GFP (PA-GFP), theyellow shifted green fluorescent protein (Yellow GFP), the yellowfluorescent protein (YFP), the enhanced yellow fluorescent protein(EYFP), the cyan fluorescent protein (CFP), the enhanced cyanfluorescent protein (ECFP), the monomeric red fluorescent protein(mRFP1), the kindling fluorescent protein (KFP1), aequorin, theautofluorescent proteins (AFPs), or the fluorescent proteins JRed,TurboGFP, PhiYFP and PhiYFP-m, tHc-Red (HcRed-Tandem), PS-CFP2 andKFP-Red (all available commercially available), or other suitablefluorescent proteins chloramphenicol acetyltransferase (CAT). Theprotein further may be selected from growth hormones, for example topromote growth in a transgenic animal, or from beta-galactosidase(lacZ), luciferase (LUC), and insulin-like growth factors (IGFs),alpha-anti-trypsin, erythropoietin (EPO), factors VIII and XI of theblood clotting system, LDL-receptor, GATA-1, etc. The nucleic acidsequence further may be a suicide gene encoding e.g. apoptotic orapoptose related enzymes and genes including A1F, Apaf e.g. Apaf-1,Apaf-2, Apaf-3, or APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2,Bcl-x.sub.L, Bcl-x.sub.S, bik, CAD, Calpain, Caspases e.g. Caspase-1,Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7,Caspase-8, Caspase-9, Caspase-10, Caspase-11, or Granzyme B, ced-3,ced-9, Ceramide, c-Jun, c-Myc, CPP32, crm A, Cytochrome c, D4-GDP-DI,Daxx, CdR1, DcR1, DD, DED, DISC, DNA-PK.sub.CS, DR3, DR4, DR5,FADD/MORT-1, FAK, Fas, Fas-ligand CD95/fas (receptor), FLICE/MACH, FLIP,Fodrin, fos, G-Actin, Gas-2, Gelsolin, glucocorticoid/glucocorticoidreceptor, granzyme A/B, hnRNPs C1/C2, ICAD, ICE, JNK, Lamin A/B, MAP,MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF-.sub.kappa.B, NuMa, p53, PAK-2,PARP, Perforin, PITSLRE, PKC.delta., pRb, Presenilin, prICE, RAIDD, Ras,RIP, Sphingomyelinase, SREBPs, thymidine kinase from Herpes simplex,TNF-.alpha., TNF-alpha receptor, TRADD, TRAF2, TRAIL-R1, TRAIL-R2,TRAIL-R3, Transglutaminase, U170 kDa snRNP, YAMA, etc.

The inventive piggyBac transposons, preferably in combination with apiggyBac transposase, has several advantages compared to approaches inthe prior art, e.g. with respect to viral and retroviral methods. Forexample, unlike proviral insertions, transposon insertions can be(re)mobilized by supplying the transposase activity in trans. Thus, forexample, instead of performing time-consuming microinjections, it ispossible according to the present invention to generate transposoninsertions at new loci.

The inventive piggyBac transposons, in combination with transposaseproteins as defined above can be transfected into a cell as a protein oras ribonucleic acid, including mRNA, as DNA, e.g. as extrachromosomalDNA including, but not limited to, episomal DNA, as plasmid DNA, or asviral nucleic acid. Furthermore, the nucleic acid encoding thetransposase protein can be transfected into a cell as a nucleic acidvector such as a plasmid, or as a gene expression vector, including aviral vector. Therefore, the nucleic acid can be circular or linear. Avector, as used herein, refers to a plasmid, a viral vector or a cosmidthat can incorporate nucleic acid encoding the transposase protein orthe transposon of this invention. The terms “coding sequence” or “openreading frame” refer to a region of nucleic acid that can be transcribedand/or translated into a polypeptide in vivo when placed under thecontrol of the appropriate regulatory sequences.

DNA encoding the transposase protein can be stably inserted into thegenome of the cell or into a vector for constitutive or inducibleexpression. Where the transposase protein is transfected into the cellor inserted into the vector as nucleic acid, the transposase encodingsequence is preferably operably linked to a promoter. There are avariety of promoters that could be used including, but not limited to,constitutive promoters, tissue-specific promoters, inducible promoters,and the like. Promoters are regulatory signals that bind RNA polymerasein a cell to initiate transcription of a downstream (3′ direction)coding sequence. A DNA sequence is operably linked to anexpression-control sequence, such as a promoter when the expressioncontrol sequence controls and regulates the transcription andtranslation of that DNA sequence. The term “operably linked” includeshaving an appropriate start signal (e.g., ATG) in front of the DNAsequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence to yield production of the desired proteinproduct. Exemplary nucleic acid sequences encoding the hyperactivepiggyBac transposon are provided as SEQ ID NO: 3-SEQ ID NO: 32 or otherhyperactive variants as described herein. In addition to theconservative changes discussed above that would necessarily alter thetransposon-encoding nucleic acid sequence (all of which are disclosedherein as well), there are other DNA or RNA sequences encoding thehyperactive piggyBac transposon protein. These DNA or RNA sequences havethe same amino acid sequence as a hyperactive piggyBac transposonprotein, but take advantage of the degeneracy of the three letter codonsused to specify a particular amino acid. For example, it is well knownin the art that various specific RNA codons (corresponding DNA codons,with a T substituted for a U) can be used interchangeably to code forspecific amino acids.

Methods for manipulating DNA and proteins are known in the art and areexplained in detail in the literature such as Sambrook et al, (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress or Ausubel, R. M., ed. (1994). Current Protocols in MolecularBiology.

Gene Transfer System

The present invention also features a gene transfer system comprising aninventive transposon, an integration defective transposon or ahyperactive piggyBac transposon as described herein, and a piggyBactransposase as described herein.

As mentioned above, the piggyBac transposase protein preferablyrecognizes repeats (e.g. IRs) on the hyperactive piggyBac transposon.The gene transfer system of this invention, therefore, preferablycomprises two components: the transposase as described herein and ahyperactive transposon or integration defective transposon as describedherein. Preferably, in certain embodiments, the transposon has at leasttwo repeats (e.g. IRs). When put together these two components provideactive transposon activity and allow the transposon to be relocated. Inuse, the transposase binds to the repeats and promotes insertion of theintervening nucleic acid sequence into DNA of a cell as defined below.

In further exemplary embodiments, the gene transfer system comprises aninventive piggyBac transposon as defined above in combination with apiggyBac transposase protein (or nucleic acid encoding a piggyBactransposase protein to provide its activity in a cell). This combinationpreferably results in the insertion of the nucleic acid sequence intothe DNA of the cell. Alternatively, it is possible to insert thetransposon of the present invention into DNA of a cell throughnon-homologous recombination through a variety of reproduciblemechanisms. In either event the inventive transposon can be used forgene transfer by using this gene transfer system.

In certain preferred embodiments, the gene transfer system mediatesinsertion of the hyperactive piggyBac transposon into the DNA of avariety of cell types and a variety of species by using the piggyBactransposase protein. Preferably, such cells include any cell suitable inthe present context, including but not limited to animal cells or cellsfrom bacteria, fungi (e.g., yeast, etc.) or plants. Preferred animalcells can be vertebrate or invertebrate. For example, preferredvertebrate cells include cells from mammals including, but not limitedto, rodents, such as rats or mice, ungulates, such as cows or goats,sheep, swine or cells from a human.

In other further exemplary embodiments, such cells, particularly cellsderived from a mammals as defined above, can be pluripotent (i.e., acell whose descendants can differentiate into several restricted celltypes, such as hematopoietic stem cells or other stem cells) andtotipotent cells (i.e., a cell whose descendants can become any celltype in an organism, e.g., embryonic stem cells). These cells areadvantageously used in order to affirm stable expression of thetransposase or to obtain a multiple number of cells already transfectedwith the components of the inventive gene transfer system. Additionally,cells such as oocytes, eggs, and one or more cells of an embryo may alsobe considered as targets for stable transfection with the present genetransfer system.

In certain preferred embodiments of the invention, the cells are stemcells.

Cells receiving the inventive piggyBac transposon and/or the piggyBactransposase protein and capable of inserting the transposon into the DNAof that cell also include without being limited thereto, lymphocytes,hepatocytes, neural cells, muscle cells, a variety of blood cells, and avariety of cells of an organism, embryonic stem cells, somatic stemcells e.g. hematopoietic cells, embryos, zygotes, sperm cells (some ofwhich are open to be manipulated by an in vitro setting).

In other certain exemplary embodiments, the cell DNA that acts as arecipient of the transposon of described herein includes any DNA presentin a cell (as mentioned above) to be transfected, if the inventivepiggyBac transposon, e.g. the hyperactive piggyBac transposon, is incontact with an piggyBac transposase protein within the cell. Forexample, the DNA can be part of the cell genome or it can beextrachromosomal, such as an episome, a plasmid, a circular or linearDNA fragment. Typical targets for insertion are e.g. double-strandedDNA.

The components of the gene transfer system described herein, i.e. thepiggyBac transposase protein (either as a protein or encoded by anucleic acid as described herein) and an inventive piggyBac transposoncan be transfected into a cell, preferably into a cell as defined above,and more preferably into the same cell. Transfection of these componentsmay furthermore occur in subsequent order or in parallel. E.g. thepiggyBac transposase protein or its encoding nucleic acid may betransfected into a cell as defined above prior to, simultaneously withor subsequent to transfection of the mammalian piggyBac transposon.Alternatively, the transposon may be transfected into a cell as definedabove prior to, simultaneously with or subsequent to transfection of thepiggyBac transposase protein or its encoding nucleic acid. Iftransfected parallel, preferably both components are provided in aseparated formulation and/or mixed with each other directly prior toadministration in order to avoid transposition prior to transfection.Additionally, administration of at least one component of the genetransfer system may occur repeatedly, e.g. by administering at leastone, two or multiple doses of this component.

For any of the above transfection reactions, the gene transfer systemmay be formulated in a suitable manner as known in the art, or as apharmaceutical composition or kit as described herein.

In further preferred embodiments, the components of the gene transfersystem may preferably be transfected into one or more cells bytechniques such as particle bombardment, electroporation,microinjection, combining the components with lipid-containing vesicles,such as cationic lipid vesicles, DNA condensing reagents (e.g., calciumphosphate, polylysine or polyethyleneimine), and inserting thecomponents (i.e. the nucleic acids thereof into a viral vector andcontacting the viral vector with the cell. Where a viral vector is used,the viral vector can include any of a variety of viral vectors known inthe art including viral vectors selected from the group consisting of aretroviral vector, an adenovirus vector or an adeno-associated viralvector.

As already mentioned above the nucleic acid encoding the piggyBactransposase protein may be RNA or DNA. Similarly, either the nucleicacid encoding the piggyBac transposase protein or the transposon of thisinvention can be transfected into the cell as a linear fragment or as acircularized fragment, preferably as a plasmid or as recombinant viralDNA.

Furthermore, the nucleic acid encoding the piggyBac transposase proteinis thereby preferably stably or transiently inserted into the genome ofthe cell to facilitate temporary or prolonged expression of the piggyBactransposase protein in the cell.

The gene transfer system as disclosed above represents a considerablerefinement of non-viral DNA-mediated gene transfer. For example,adapting viruses as agents for gene therapy restricts genetic design tothe constraints of that virus genome in terms of size, structure andregulation of expression. Non-viral vectors, as described herein, aregenerated largely from synthetic starting materials and are thereforemore easily manufactured than viral vectors. Non-viral reagents are lesslikely to be immunogenic than viral agents making repeat administrationpossible. Non-viral vectors are more stable than viral vectors andtherefore better suited for pharmaceutical formulation and applicationthan are viral vectors. Additionally, the inventive gene transfer systemis a non-viral gene transfer system that facilitates insertion into DNAand markedly improves the frequency of stable gene transfer.

The present invention further provides an efficient method for producingtransgenic animals, including the step of applying the inventive genetransfer system to an animal. Transgenic DNA has not been efficientlyinserted into chromosomes. Only about one in a million of the foreignDNA molecules is inserted into the cellular genome, generally severalcleavage cycles into development. Consequently, most transgenic animalsare mosaic (Hackett et al. (1993). The molecular biology of transgenicfish. In Biochemistry and Molecular Biology of Fishes (Hochachka &Mommsen, eds) Vol. 2, pp. 207-240). As a result, animals raised fromembryos into which transgenic DNA has been delivered must be cultureduntil gametes can be assayed for the presence of inserted foreign DNA.Many transgenic animals fail to express the transgene due to positioneffects. A simple, reliable procedure that directs early insertion ofexogenous DNA into the chromosomes of animals at the one-cell stage isneeded. The present system helps to fill this need.

In certain preferred embodiments, the gene transfer system of thisinvention can readily be used to produce transgenic animals that carry aparticular marker or express a particular protein in one or more cellsof the animal. Generally, methods for producing transgenic animals areknown in the art and incorporation of the inventive gene transfer systeminto these techniques does not require undue experimentation, e.g. thereare a variety of methods for producing transgenic animals for researchor for protein production including, but not limited to Hackett et al.(1993, supra). Other methods for producing transgenic animals aredescribed in the art (e.g. M. Markkula et al. Rev. Reprod., 1, 97-106(1996); R. T. Wall et al., J. Dairy Sci., 80, 2213-2224 (1997)), J. C.Dalton, et al. (Adv. Exp. Med. Biol., 411, 419-428 (1997)) and H. Lubonet al. (Transfus. Med. Rev., 10, 131-143 (1996)).

In another embodiment, the present invention features a transgenicanimal produced by the methods described herein, preferably by using thegene transfer system presently described. For example, transgenicanimals may preferably contain a nucleic acid sequence inserted into thegenome of the animal by the gene transfer system, thereby enabling thetransgenic animal to produce its gene product, e.g. a protein. Intransgenic animals this protein is preferably a product for isolationfrom a cell, for example the inventive protein can be produced inquantity in milk, urine, blood or eggs. Promoters can be used thatpromote expression in milk, urine, blood or eggs and these promotersinclude, but are not limited to, casein promoter, the mouse urinaryprotein promoter, beta-globin promoter and the ovalbumin promoterrespectively. Recombinant growth hormone, recombinant insulin, and avariety of other recombinant proteins have been produced using othermethods for producing protein in a cell. Nucleic acids encoding these orother proteins can be inserted into the transposon of this invention andtransfected into a cell. Efficient transfection of the inventivetransposon as defined above into the DNA of a cell occurs when mammalianpiggyBac transposase protein is present. Where the cell is part of atissue or part of a transgenic animal, large amounts of recombinantprotein can be obtained.

Transgenic animals may be selected from vertebrates and invertebrates,e.g. fish, birds, mammals including, but not limited to, rodents, suchas rats or mice, ungulates, such as cows or goats, sheep, swine orhumans.

The present invention furthermore provides a method for gene therapycomprising the step of introducing the gene transfer system into cellsas described herein. Therefore, the inventive piggyBac transposons asdescribed herein preferably comprises a gene to provide a gene therapyto a cell or an organism. Preferably, the gene is placed under thecontrol of a tissue specific promoter or of a ubiquitous promoter or oneor more other expression control regions for the expression of a gene ina cell in need of that gene. Presently, a variety of genes are beingtested for a variety of gene therapies including, but not limited to,the CFTR gene for cystic fibrosis, adenosine deaminase (ADA) for immunesystem disorders, factor IX and interleukin-2 (IL-2) for blood celldiseases, alpha-1-antitrypsin for lung disease, and tumor necrosisfactors (INFs) and multiple drug resistance (MDR) proteins for cancertherapies. These and a variety of human or animal specific genesequences including gene sequences to encode marker proteins and avariety of recombinant proteins are available in the known genedatabases such as GenBank.

An advantage of the inventive gene transfer system for gene therapypurposes is that it is not limited to a great extent by the size of theintervening nucleic acid sequence positioned between the repeats. Thereis no known limit on the size of the nucleic acid sequence that can beinserted into DNA of a cell using the mammalian piggyBac transposaseprotein.

In particular preferred embodiments, for gene therapy purposes, but alsofor other inventive purposes the gene transfer system may be transfectedinto cells by a variety of methods, e.g. by microinjection,lipid-mediated strategies or by viral-mediated strategies. For example,where microinjection is used, there is very little restraint on the sizeof the intervening sequence of the transposon of this invention.Similarly, lipid-mediated strategies do not have substantial sizelimitations. However, other strategies for introducing the gene transfersystem into a cell, such as viral-mediated strategies could limit thelength of the nucleic acid sequence positioned between the repeats.

Accordingly, in certain exemplary embodiments, the gene transfer systemas described herein can be delivered to cells via viruses, includingretroviruses (such as lentiviruses, etc.), adenoviruses,adeno-associated viruses, herpes viruses, and others. There are severalpotential combinations of delivery mechanisms that are possible for thehyperactive piggyBac transposon portion containing the transgene ofinterest flanked by the terminal repeats and the gene encoding thetransposase. For example, both the transposon and the transposase genecan be contained together on the same recombinant viral genome; a singleinfection delivers both parts of the gene transfer system such thatexpression of the transposase then directs cleavage of the transposonfrom the recombinant viral genome for subsequent insertion into acellular chromosome. In another example, the transposase and thetransposon can be delivered separately by a combination of virusesand/or non-viral systems such as lipid-containing reagents. In thesecases either the transposon and/or the transposase gene can be deliveredby a recombinant virus. In every case, the expressed transposase genedirects liberation of the transposon from its carrier DNA (viral genome)for insertion into chromosomal DNA.

In certain preferred embodiments of the present invention, inventivepiggyBac transposons may be utilized for insertional mutagenesis,preferably followed by identification of the mutated gene. DNAtransposons, particularly the transposons, have several advantagescompared to approaches in the prior art, e.g. with respect to viral andretroviral methods. For example, unlike proviral insertions, transposoninsertions can be remobilized by supplying the transposase activity intrans. Thus, instead of performing time-consuming microinjections, it ispossible according to the present invention to generate transposoninsertions at new loci by crossing stocks transgenic for the abovementioned two components of the transposon system, the inventivetransposon and the inventive transposase. In a preferred embodiment thegene transfer system is directed to the germline of the experimentalanimals in order to mutagenize germ cells. Alternatively, transposaseexpression can be directed to particular tissues or organs by using avariety of specific promoters. In addition, remobilization of amutagenic transposon out of its insertion site can be used to isolaterevertants and, if transposon excision is associated with a deletion offlanking DNA, the inventive gene transfer system may be used to generatedeletion mutations. Furthermore, since transposons are composed of DNA,and can be maintained in simple plasmids, inventive transposons andparticularly the use of the inventive gene transfer system is much saferand easier to work with than highly infectious retroviruses. Thetransposase activity can be supplied in the form of DNA, mRNA or proteinas defined above in the desired experimental phase.

In another embodiment, the present invention also provides an efficientsystem for gene discovery, e.g. genome mapping, by introducing aninventive piggyBac transposon, as defined above into a gene using a genetransfer system as described in the present invention. In one example,the hyperactive piggyBac transposon in combination with the piggyBactransposase protein or a nucleic acid encoding the piggyBac transposaseprotein is transfected into a cell. In certain preferred embodiments,the transposon preferably comprises a nucleic acid sequence positionedbetween at least two repeats, wherein the repeats bind to the piggyBactransposase protein and wherein the transposon is inserted into the DNAof the cell in the presence of the piggyBac transposase protein. Incertain preferred embodiments, the nucleic acid sequence includes amarker protein, such as GFP and a restriction endonuclease recognitionsite. Following insertion, the cell DNA is isolated and digested withthe restriction endonuclease. For example, if the endonucleaserecognition site is a 6-base recognition site and a restrictionendonuclease is used that employs a 6-base recognition sequence, thecell DNA is cut into about 4000-bp fragments on average. These fragmentscan be either cloned or linkers can be added to the ends of the digestedfragments to provide complementary sequence for PCR primers. Wherelinkers are added, PCR reactions are used to amplify fragments usingprimers from the linkers and primers binding to the direct repeats ofthe repeats in the transposon. The amplified fragments are thensequenced and the DNA flanking the direct repeats is used to searchcomputer databases such as GenBank.

Using the gene transfer system for methods as disclosed above such asgene discovery and/or gene tagging, permits, for example,identification, isolation, and characterization of genes involved withgrowth and development through the use of transposons as insertionalmutagens or identification, isolation and characterization oftranscriptional regulatory sequences controlling growth and development.

In another exemplary embodiment of the present invention, the inventionprovides a method for mobilizing a nucleic acid sequence in a cell.According to this method the hyperactive piggyBac transposon is insertedinto DNA of a cell, as described herein. Hyperactive piggyBac protein ornucleic acid encoding the piggyBac transposase protein is transfectedinto the cell and the protein is able to mobilize (i.e. move) thetransposon from a first position within the DNA of the cell to a secondposition within the DNA of the cell. The DNA of the cell is preferablygenomic DNA or extrachromosomal DNA. The inventive method allowsmovement of the transposon from one location in the genome to anotherlocation in the genome, or for example, from a plasmid in a cell to thegenome of that cell.

In another exemplary embodiments, the inventive gene transfer system canalso be used as part of a method involving RNA-interference techniques.RNA interference (RNAi), is a technique in which exogenous,double-stranded RNAs (dsRNAs), being complementary to mRNA's orgenes/gene fragments of the cell, are introduced into this cell tospecifically bind to a particular mRNA and/or a gene and therebydiminishing or abolishing gene expression. The technique has proveneffective in Drosophila, Caenorhabditis elegans, plants, and recently,in mammalian cell cultures. In order to apply this technique in contextwith the present invention, the inventive transposon preferably containsshort hairpin expression cassettes encoding small interfering RNAs(siRNAs), which are complementary to mRNA's and/or genes/gene fragmentsof the cell. These siRNAs have preferably a length of 20 to 30 nucleicacids, more preferably a length of 20 to 25 nucleic acids and mostpreferably a length of 21 to 23 nucleic acids. The siRNA may be directedto any mRNA and/or a gene, that encodes any protein as defined above,e.g. an oncogene. This use, particularly the use of mammalian piggyBactransposons for integration of siRNA vectors into the host genomeprovides a long-term expression of siRNA in vitro or in vivo and thusenables a long-term silencing of specific gene products.

Induced Pluripotent Stem Cells (iPS)

In certain preferred embodiments, the present invention may include areprogramming vector that includes a polycistronic expression cassettecomprising a transcriptional regulatory element, one or morereprogramming factors, and one or more hyperactive piggyBac transposonsas described herein. Preferably, the reprogramming factor encoded isSox, Oct, Nanog, Klf4, or c-Myc

In general, stem cells are undifferentiated cells which can give rise toa succession of mature functional cells. For example, a hematopoieticstem cell may give rise to any of the different types of terminallydifferentiated blood cells. Embryonic stem (ES) cells are derived fromthe embryo and are pluripotent, thus possessing the capability ofdeveloping into any organ or tissue type or, at least potentially, intoa complete embryo.

Induced pluripotent stem cells, commonly abbreviated as iPS cells oriPSCs, are a type of pluripotent stem cells artificially derived fromnon-pluripotent cells, typically adult somatic cells, by insertingcertain genes. Induced pluripotent stem cells are believed to beidentical to natural pluripotent stem cells, such as embryonic stemcells in many respects, for example, in the expression of certain stemcell genes and proteins, chromatin methylation patterns, doubling time,embryoid body formation, teratoma formation, viable chimera formation,and potency and differentiability, but the full extent of their relationto natural pluripotent stem cells is still being assessed.

iPS cells were first produced in 2006 (Takahashi et al., 2006,incorporated by reference in its entirety herein) from mouse cells andin 2007 from human cells (Takahashi et al., 2007, incorporated byreference in its entirety herein). This has been cited as an importantadvancement in stem cell research, as it may allow researchers to obtainpluripotent stem cells, which are important in research and potentiallyhave therapeutic uses, without the controversial use of embryos.

“Reprogramming” is a process that confers on a cell a measurablyincreased capacity to form progeny of at least one new cell type, eitherin culture or in vivo, than it would have under the same conditionswithout reprogramming More specifically, reprogramming is a process thatconfers on a somatic cell a pluripotent potential. This means that aftersufficient proliferation, a measurable proportion of progeny havingphenotypic characteristics of the new cell type if essentially no suchprogeny could form before reprogramming; otherwise, the proportionhaving characteristics of the new cell type is measurably more thanbefore reprogramming Under certain conditions, the proportion of progenywith characteristics of the new cell type may be at least about 1%, 5%,25% or more in the in order of increasing preference.

Embryonic stem (ES) cells” are pluripotent stem cells derived from earlyembryos. An ES cell was first established in 1981, which has also beenapplied to production of knockout mice since 1989. In 1998, a human EScell was established, which is currently becoming available forregenerative medicine.

Unlike ES cells, tissue stem cells have a limited differentiationpotential. Tissue stem cells are present at particular locations intissues and have an undifferentiated intracellular structure. Therefore,the pluripotency of tissue stem cells is typically low. Tissue stemcells have a higher nucleus/cytoplasm ratio and have few intracellularorganelles. Most tissue stem cells have low pluripotency, a long cellcycle, and proliferative ability beyond the life of the individual.Tissue stem cells are separated into categories, based on the sites fromwhich the cells are derived, such as the dermal system, the digestivesystem, the bone marrow system, the nervous system, and the like. Tissuestem cells in the dermal system include epidermal stem cells, hairfollicle stem cells, and the like. Tissue stem cells in the digestivesystem include pancreatic (common) stem cells, liver stem cells, and thelike. Tissue stem cells in the bone marrow system include hematopoieticstem cells, mesenchymal stem cells, and the like. Tissue stem cells inthe nervous system include neural stem cells, retinal stem cells, andthe like.

“Induced pluripotent stem cells,” commonly abbreviated as iPS cells oriPSCs, refer to a type of pluripotent stem cell artificially preparedfrom a non-pluripotent cell, typically an adult somatic cell, orterminally differentiated cell, such as fibroblast, a hematopoieticcell, a myocyte, a neuron, an epidermal cell, or the like, by insertingcertain genes, referred to as reprogramming factors.

The generation of iPS cells is crucial on the genes used for theinduction. The following factors or combination thereof could be used inthe present invention. In certain aspects, nucleic acids encoding Soxand Oct (preferably Oct3/4) will be included into the reprogrammingvector. For example, a reprogramming vector may comprise expressioncassettes encoding Sox2, Oct4, Nanog and optionally Lin-28, orexpression cassettes encoding Sox2, Oct4, Klf4 and optionally c-myc.Nucleic acids encoding these reprogramming factors may be comprised inthe same expression cassette, different expression cassettes, the samereprogramming vector, or different reprogramming vectors.

Oct-3/4 and certain members of the Sox gene family (Sox1, Sox2, Sox3,and Sox15) have been identified as crucial transcriptional regulatorsinvolved in the induction process whose absence makes inductionimpossible. Additional genes, however, including certain members of theKlf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (C-myc, L-myc,and N-myc), Nanog, and LIN28, have been identified to increase theinduction efficiency. Oct-3/4 (Pou5fl) is one of the family of octamer(“Oct”) transcription factors, and plays a crucial role in maintainingpluripotency. The absence of Oct-3/4 in Oct-3/4+ cells, such asblastomeres and embryonic stem cells, leads to spontaneous trophoblastdifferentiation, and presence of Oct-3/4 thus gives rise to thepluripotency and differentiation potential of embryonic stem cells.Various other genes in the “Oct” family, including Oct-3/4's closerelatives, Oct1 and Oct6, fail to elicit induction, thus demonstratingthe exclusiveness of Oct-3/4 to the induction process.

The Sox family of genes is associated with maintaining pluripotencysimilar to Oct-3/4, although it is associated with multipotent andunipotent stem cells in contrast with Oct-3/4, which is exclusivelyexpressed in pluripotent stem cells. While Sox2 was the initial geneused for induction by Yamanaka et al. (2007), Jaenisch et al. (1988) andYu et al. (2007), other genes in the Sox family have been found to workas well in the induction process. Sox1 yields iPS cells with a similarefficiency as Sox2, and genes Sox3, Sox15, and Sox18 also generate iPScells, although with decreased efficiency.

In embryonic stem cells, at least an Oct member such as Oct-3/4 and atleast a Sox member such as Sox2, are necessary in promotingpluripotency. Yamanaka et al. (2007) reported that Nanog was unnecessaryfor induction although Yu et al. (2007) has reported it is possible togenerate iPS cells with Nanog as one of the factors and Nanog certainlyenhances reprogramming efficiency dose-dependently.

Klf4 of the Klf family of genes was initially identified by Yamanaka etal. and confirmed by Jaenisch et al. (1988) as a factor for thegeneration of mouse iPS cells and was demonstrated by Yamanaka et al.(2007) as a factor for generation of human iPS cells. However, Thompsonet al. reported that Klf4 was unnecessary for generation of human iPScells and in fact failed to generate human iPS cells. Klf2 and Klf4 werefound to be factors capable of generating iPS cells, and related genesKlf1 and Klf5 did as well, although with reduced efficiency.

The Myc family of genes are proto-oncogenes implicated in cancer.Yamanaka et al. and Jaenisch et al. (1988) demonstrated that c-myc is afactor implicated in the generation of mouse iPS cells and Yamanaka etal. demonstrated it was a factor implicated in the generation of humaniPS cells. However, Thomson et al. and Yamanaka et al. (2007) reportedthat c-myc was unnecessary for generation of human iPS cells. Usage ofthe “myc” family of genes in induction of iPS cells is troubling for theeventuality of iPS cells as clinical therapies, as 25% of micetransplanted with c-myc-induced iPS cells developed lethal teratomas.N-myc and L-myc have been identified to induce in the stead of c-mycwith similar efficiency.

Pharmaceutical Compositions

The present invention further refers to pharmaceutical compositionscontaining either a piggyBac transposase as a protein or encoded by anucleic acid, and/or a hyperactive piggyBac transposon, or a genetransfer system as described herein comprising a piggyBac transposase asa protein or encoded by a nucleic acid, in combination with ahyperactive piggyBac transposon.

The pharmaceutical composition may optionally be provided together witha pharmaceutically acceptable carrier, adjuvant or vehicle. In thiscontext, a pharmaceutically acceptable carrier, adjuvant, or vehicleaccording to the invention refers to a non-toxic carrier, adjuvant orvehicle that does not destroy the pharmacological activity of thecomponent(s) with which it is formulated. Pharmaceutically acceptablecarriers, adjuvants or vehicles that may be used in the compositions ofthis invention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.

The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the pharmaceutical compositions areadministered orally, intraperitoneally or intravenously. Sterileinjectable forms of the pharmaceutical compositions of this inventionmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the inventive genetransfer system or components thereof with a suitable non-irritatingexcipient that is solid at room temperature but liquid at rectaltemperature and Therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the inventive genetransfer system or components thereof suspended or dissolved in one ormore carriers. Carriers for topical administration of the components ofthis invention include, but are not limited to, mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene component, emulsifying wax and water. Alternatively,the pharmaceutically acceptable compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of the components of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. It has to be noted that a specific dosage andtreatment regimen for any particular patient will depend upon a varietyof factors, including the activity of the specific component employed,the age, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a component of the present invention in the composition willalso depend upon the particular component(s) in the composition.

The pharmaceutical composition is preferably suitable for the treatmentof diseases, particular diseases caused by gene defects such as cysticfibrosis, hypercholesterolemia, hemophilia, immune deficienciesincluding HIV, Huntington disease, .alpha.-anti-Trypsin deficiency, aswell as cancer selected from colon cancer, melanomas, kidney cancer,lymphoma, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL),chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL),gastrointestinal tumors, lung cancer, gliomas, thyroid cancer, mammacarcinomas, prostate tumors, hepatomas, diverse virus-induced tumorssuch as e.g. papilloma virus induced carcinomas (e.g. cervix carcinoma),adeno carcinomas, herpes virus induced tumors (e.g. Burkitt's lymphoma,EBV induced B cell lymphoma), Hepatitis B induced tumors (Hepato cellcarcinomas), HTLV-1 and HTLV-2 induced lymphoma, lung cancer, pharyngealcancer, anal carcinoma, glioblastoma, lymphoma, rectum carcinoma,astrocytoma, brain tumors, stomach cancer, retinoblastoma, basalioma,brain metastases, medullo blastoma, vaginal cancer, pancreatic cancer,testis cancer, melanoma, bladder cancer, Hodgkin syndrome, meningeoma,Schneeberger's disease, bronchial carcinoma, pituitary cancer, mycosisfungoides, gullet cancer, breast cancer, neurinoma, spinalioma,Burkitt's lymphoma, laryngeal cancer, thymoma, corpus carcinoma, bonecancer, non-Hodgkin lymphoma, urethra cancer, CUP-syndrome,oligodendroglioma, vulva cancer, intestinal cancer, oesphagus carcinoma,small intestine tumors, craniopharyngeoma, ovarial carcinoma, ovariancancer, liver cancer, leukemia, or cancers of the skin or the eye; etc.

Kits

The present invention also features kits comprising a piggyBactransposase as a protein or encoded by a nucleic acid, and/or ahyperactive piggyBac transposon; or a gene transfer system as describedherein comprising a piggyBac transposase as a protein or encoded by anucleic acid as described herein, in combination with a hyperactivepiggyBac transposon; optionally together with a pharmaceuticallyacceptable carrier, adjuvant or vehicle, and optionally withinstructions for use.

Any of the components of the inventive kit may be administered and/ortransfected into cells in a subsequent order or in parallel. e.g. thepiggyBac transposase protein or its encoding nucleic acid may beadministered and/or transfected into a cell as defined above prior to,simultaneously with or subsequent to administration and/or transfectionof the inventive hyperactive transposon. Alternatively, the hyperactivepiggyBac transposon may be transfected into a cell as defined aboveprior to, simultaneously with or subsequent to transfection of thepiggyBac transposase protein or its encoding nucleic acid. Iftransfected parallel, preferably both components are provided in aseparated formulation and/or mixed with each other directly prior toadministration in order to avoid transposition prior to transfection.Additionally, administration and/or transfection of at least onecomponent of the kit may occur in a time staggered mode, e.g. byadministering multiple doses of this component.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 Identification of Integration-Defective piggyBacVariants

The present experiments describe screening and identification ofexcision-hyperactive piggyBac transposons using a version of the Cherrygene which produces a red fluorescent protein. A copy of the piggyBactransposon was put into the gene, inactivating it such as the cells areNOT red. However, piggyBac excision restores the gene, leading to theproduction of the red fluorescent protein. Accordingly, increased redcolony color identifies mutants that excise better.

A large collection of mutant transposase genes was made using mutagenicPCR which was cloned into an expression vector in yeast. Coloniescontaining individual mutants were grow up on an agar plate and werethen examined with red fluorescent light. FIG. 4 shows excisionhyperactives that have been isolated to date.

In certain preferred embodiments, the integration defective piggyBaccomprises an amino acid change in the wild type piggyBac sequencecorresponding to SEQ ID NO: 2. Preferably, the amino acid change isR371A, R373A or R371A, R373A.

In preferred exemplary embodiments, the integration defective piggyBaccorresponds to the amino acid sequence set forth as SEQ ID NO: 64, SEQID NO: 65 or SEQ ID NO: 66.

Example 2 Identification of Hyperactive Variants

Using the integration defective piggyBac mutants as a starting point,the present inventors have identified hyperactive piggyBac transposonmutants. The yeast excision assay that was developed as described inMitra R. et al. (piggyBac can bypass DNA synthesis during cut and pastetransposition. EMBO J. April 9; 27(7):1097-109. Epub 2008 Mar. 20) wasused to identify the hyperactive mutants. The piggyBac ORF wasmutagenized by mutagenic PCR using primers flanking the ORF as theexpression construct and then recovered transformants byco-transformation of the PCR product with a gapped piggyBac plasmid intothe yeast assay strain containing a ura− to ura+ cassesette in whichtransposon excision results in formation of ura+ colonies. Followingrecovery of transformants on SC-Trp-His plates, colonies wereresuspended in water and spotted onto plates lacking uracil to identifyexcisions. By comparison to the number of ura+ colonies from themutagenized transformants to wildtype in these spotting tests, potentialhyperactive variants were identified. Each hyperactive candidate strainwas then purified and quantitatively reassayed excision. Plasmid DNAcontaining the piggyBac gene from confirmed hyperactives was thensequenced to identify the piggyBac gene mutation and resulting aminoacid change Amino acid changes and corresponding nucleic acid changesare shown in Table 1, below:

TABLE 1 L15P CUG to CCG D19N/F395L GAC to AAC/UUU to CUU S31P/T164AUCA to CCA/ACA to GCA H33Y CAC to UAC E44K/K334R GAA to AAA/AAG to AGGE45G GAA to GGA C97R/T242I UGU to CGU/ACU to AUU S103P UCC to CCCR189K/G120G AGA to AAA/GGU to GGC R189R/D450N/R526RAGA to AGG/GAC to AAC M194T AUG to ACG M194V AUG to GUG S213S/V436IAGU to AGC I221T AUA to ACA S373P between M6+ UCA to CCA N384TAAC to ACC C453S/N571S UGU to AGU/AAU to AGU T560A ACU to GCU N571SAAU to AAG S573A UCG to GCG S584P UCU to CCU M589V AUG to GUGM589V/D170D ATG to GUG/GAC to GAU S592G AGU to GGU F594L UUC to TTAStop/WLESCN TGA to TGG Stop595ELESCN/H33H TGA to GGA/CAC to CAUTable 1 discloses “WLESCN” as SEQ ID NO: 128 and “ELESCN” as SEQ ID NO:129.

The amino acid changes and fold increase in transposition from that ofwildtype (normalized to 1) for certain exemplary hyperactive mutants isshown in Table 2 in FIG. 1.

Hyperactive mutations can occur at many positions within the transposaseand it is expected that many more hyperactive piggyBac variants will befound. These variants may be altered in a single amino acid or multipleamino acids. Variants can be identified using the yeast assay as ascreen. PCR mutagenesis of the entire gene as well as targetedmutagenesis using smaller piggyBac fragments or oligonucleotide-directedmutagenesis to regions that have been identified as giving hyperactivemutations will be used.

Example 3 Induced Pluripotent Stem Cell Generation Using the HyperactiveTransposon

In certain exemplary embodiments, the hyperactive piggyBac transposonscan be used to created induced pluripotent stem cells using a minimalset of genes. In particular, Oct ¾, Sox2, Klf4 and c-myc are used as aminimal set of genes. Takahashi et al. (Cell, 131, 861-872, Nov. 30,2007), incorporated by reference in its entirety herein, teach methodsof generating induced pluripotent stem cells (iPS) from human dermalfibroblasts using Oct 3/4, Sox2, klf4, and c-Myc.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A transposon comprising: one or more hyperactivepiggyBac nucleic acid sequences and variants, derivatives and fragmentsthereof that retain transposon activity.
 2. The transposon of claim 1,wherein the hyperactive piggyBac transposon has a higher level oftransposon excision compared to a wildtype piggyBac transposon.
 3. Thetransposon of claim 1, comprising 2, 3, 4, 5 or more hyperactivepiggyBac nucleic acid sequences and variants, derivatives and fragmentsthereof that retain transposon activity.
 4. The transposon of claim 1,wherein the hyperactive piggyBac nucleic acid sequence is from thefamily Noctuidae.
 5. The transposon of claim 1, wherein the hyperactivepiggyBac nucleic acid sequence is from the species Trichoplusia ni. 6.The transposon of claim 1, comprising a nucleic acid sequence selectedfrom the group consisting of: SEQ ID NO: 34-SEQ ID NO: 63 or SEQ ID NO:70 SEQ ID NO:
 96. 7. The transposon of claim 6, wherein the nucleic acidsequence encodes an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 3-SEQ ID NO:
 32. 8. A transposon comprising:one or more integration defective piggyBac nucleic acid sequences andvariants, derivatives and fragments thereof.
 9. The transposon of claim8, wherein the integration defective piggyBac transposon has a lowerrate of integration as compared to a wildtype piggyBac transposon. 10.The transposon of claim 8, wherein the integration defective piggyBacnucleic acid sequence is from the family Noctuidae.
 11. The transposonof claim 8, wherein the integration defective piggyBac nucleic acidsequence is from the species Trichoplusia ni.
 12. The transposon ofclaim 8, comprising a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO: 67-SEQ ID NO:
 69. 13. The transposon of claim12, wherein the nucleic acid sequence encodes an amino acid sequenceselected from the group consisting of: SEQ ID NO: 64-SEQ ID NO:
 66. 14.The transposon of claim 2 or claim 9, wherein the wildtype piggyBactransposon comprises a nucleic acid sequence corresponding to SEQ IDNO:
 1. 15. The transposon of claim 1 or claim 8, wherein the transposonis capable of inserting into the DNA of a cell.
 16. The transposon ofclaim 1 or claim 8, wherein the transposon further comprises a markerprotein.
 17. The transposon of claim 1 or claim 8, wherein thetransposon is inserted in a plasmid.
 18. The transposon of claim 17wherein the transposon further comprises at least a portion of an openreading frame.
 19. The transposon of claim 17 wherein the transposonfurther comprises at least one expression control region.
 20. Thetransposon of claim 17 wherein the expression control region is selectedfrom the group consisting of a promoter, an enhancer or a silencer. 21.The transposon of claim 17 wherein the transposon further comprises apromoter operably linked to at least a portion of an open reading frame.22. The transposon of claim 15 wherein the cell is obtained from ananimal.
 23. The transposon of claim 22 wherein the cell is from avertebrate or an invertebrate.
 24. The transposon of claim 17 whereinthe vertebrate is a mammal.
 25. A gene transfer system comprising: atransposon according to claim 1 or claim 8; and a piggyBac transposase.26. The gene transfer system of claim 25, wherein the piggyBactransposase is from the family Noctuidae.
 27. The gene transfer systemof claim 26, wherein the piggyBac transposase is from the speciesTrichoplusia ni.
 28. The gene transferase system of claim 27, whereinthe piggyBac transposase comprises an amino acid sequence correspondingto SEQ ID NO:
 33. 29. The gene transfer system of claim 25, wherein thepiggyBac transposase is a mammalian piggyBac transposase.
 30. The genetransfer system of claim 25 wherein the transposon is inserted into thegenome of the cell.
 31. The gene transfer system of claim 30, whereinthe cell is obtained from an animal.
 32. The gene transfer system ofclaim 30, wherein the cell is from a vertebrate or an invertebrate. 33.The gene transfer system of claim 32, wherein the vertebrate is amammal.
 34. A cell comprising the transposon of claim
 1. 35. A cellcomprising the transposon of claim
 8. 36. A pharmaceutical compositioncomprising: a transposon comprising a hyperactive piggyBac nucleic acidsequence and a piggyBac transposase, together with a pharmaceuticallyacceptable carrier, adjuvant or vehicle.
 37. A method for introducingexogenous DNA into a cell comprising: contacting the cell with the genetransfer system of claim 25, thereby introducing exogenous DNA into acell.
 38. The method of claim 37, wherein the cell is a stem cell.
 39. Akit comprising: a transposon comprising a hyperactive piggyBac nucleicacid sequence and instructions for introducing DNA into a cell.
 40. Thekit of claim 39, wherein the hyperactive piggyBac nucleic acid sequenceis from the family Noctuidae.
 41. The kit of claim 39, wherein thehyperactive piggyBac nucleic acid sequence is from the speciesTrichoplusia ni.
 42. The kit of claim 39, comprising a nucleic acidsequence selected from the group consisting of: SEQ ID NO: 34-SEQ ID NO:63 or SEQ ID NO: 70-SEQ ID NO:
 96. 43. A kit comprising: a transposoncomprising a integration defective piggyBac nucleic acid sequence andinstructions for use.
 44. The kit of claim 43, comprising a nucleic acidsequence selected from the group consisting of: SEQ ID NO: 67-SEQ ID NO:69.